Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines.

Mentions:
HIPSTR levels do not correlate with the expression of TFAP2A. We then reasoned that chromatin-associated HIPSTR lncRNA might regulate other genes elsewhere in the genome in trans. Consistent with a relatively short half-life of this lncRNA (38 min, Fig. S3A), efficient HIPSTR silencing in HEK293 cells with a pool of targeting ASOs was achieved as early as 6 h after transfection (~71%, Fig. S3B), and the highest efficiency was reached 24 h post-transfection (~89%, Fig. S3B). HIPSTR silencing in HEK293 cells with each of the two targeting ASOs separately (ASO #1 and ASO #2; Fig. 3A) did not affect the overall levels of TFAP2A expression (Fig. 3A), but instead it significantly altered TFAP2A-AS1 expression (Fig. 3A; see further details below). Neither the mRNA levels of specific TFAP2A isoforms, nor the levels of TFAP2A pre-mRNA were affected (Fig. 3B). HIPSTR knockdown resulted in genome-wide differential expression of 380 annotated genes (439 probes) located outside of the TFAP2A locus (Fig. 3C, Table S1), of which 377 (~99.2%) were upregulated. These results suggest a repressive function for HIPSTR in HEK293 cells. Accordingly, transient overexpression of HIPSTR in HEK293 cells (Fig. S3C) resulted in downregulation (Fig. S3D) of eight out of the twelve selected genes that were upregulated in HIPSTR knockdown experiments (Table S1). At the same time, Gene Ontology (GO) analysis of the genes upregulated by HIPSTR knockdown revealed their enrichment in “Developmental Process” and “Cell Differentiation” categories (Fig. S3E). A group of genes upregulated by HIPSTR knockdown in HEK293 cells was also upregulated by HIPSTR silencing in LNCaP cells (Fig. S3F), further validating these results.

Mentions:
HIPSTR levels do not correlate with the expression of TFAP2A. We then reasoned that chromatin-associated HIPSTR lncRNA might regulate other genes elsewhere in the genome in trans. Consistent with a relatively short half-life of this lncRNA (38 min, Fig. S3A), efficient HIPSTR silencing in HEK293 cells with a pool of targeting ASOs was achieved as early as 6 h after transfection (~71%, Fig. S3B), and the highest efficiency was reached 24 h post-transfection (~89%, Fig. S3B). HIPSTR silencing in HEK293 cells with each of the two targeting ASOs separately (ASO #1 and ASO #2; Fig. 3A) did not affect the overall levels of TFAP2A expression (Fig. 3A), but instead it significantly altered TFAP2A-AS1 expression (Fig. 3A; see further details below). Neither the mRNA levels of specific TFAP2A isoforms, nor the levels of TFAP2A pre-mRNA were affected (Fig. 3B). HIPSTR knockdown resulted in genome-wide differential expression of 380 annotated genes (439 probes) located outside of the TFAP2A locus (Fig. 3C, Table S1), of which 377 (~99.2%) were upregulated. These results suggest a repressive function for HIPSTR in HEK293 cells. Accordingly, transient overexpression of HIPSTR in HEK293 cells (Fig. S3C) resulted in downregulation (Fig. S3D) of eight out of the twelve selected genes that were upregulated in HIPSTR knockdown experiments (Table S1). At the same time, Gene Ontology (GO) analysis of the genes upregulated by HIPSTR knockdown revealed their enrichment in “Developmental Process” and “Cell Differentiation” categories (Fig. S3E). A group of genes upregulated by HIPSTR knockdown in HEK293 cells was also upregulated by HIPSTR silencing in LNCaP cells (Fig. S3F), further validating these results.

Eukaryotic genomes are transcribed into numerous regulatory long non-coding RNAs (lncRNAs). Compared to mRNAs, lncRNAs display higher developmental stage-, tissue-, and cell-subtype-specificity of expression, and are generally less abundant in a population of cells. Despite the progress in single-cell-focused research, the origins of low population-level expression of lncRNAs in homogeneous populations of cells are poorly understood. Here, we identify HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel lncRNA gene in the developmentally regulated TFAP2A locus. HIPSTR has evolutionarily conserved expression patterns, its promoter is most active in undifferentiated cells, and depletion of HIPSTR in HEK293 and in pluripotent H1BP cells predominantly affects the genes involved in early organismal development and cell differentiation. Most importantly, we find that HIPSTR is specifically induced and heterogeneously expressed in the 8-cell-stage human embryos during the major wave of embryonic genome activation. We systematically explore the phenomenon of cell-to-cell variation of gene expression and link it to low population-level expression of lncRNAs, showing that, similar to HIPSTR, the expression of thousands of lncRNAs is more highly heterogeneous than the expression of mRNAs in the individual, otherwise indistinguishable cells of totipotent human embryos, primordial germ cells, and stable cell lines.